Báo cáo y học: "Identification of target antigens of antiendothelial cell and anti-vascular smooth muscle cell antibodies in patients with giant cell arteritis: a proteomic approach" potx

Régent et al Arthritis Research & Therapy 2011, 13:R107 http://arthritis-research.com/content/13/3/R107 RESEARCH ARTICLE Open Access Identification of target antigens of antiendothelial cell and anti-vascular smooth muscle cell antibodies in patients with giant cell arteritis: a proteomic approach Alexis Régent1,2,3, Hanadi Dib1,2, Kim H Ly1,2,4†, Christian Agard5†, Mathieu C Tamby1,2, Nicolas Tamas1,2, Babette Weksler6, Christian Federici1,2, Cédric Broussard7, Loïc Guillevin2,3 and Luc Mouthon1,2,3* Abstract Introduction: Immunological studies of giant cell arteritis (GCA) suggest that a triggering antigen of unknown nature could generate a specific immune response We thus decided to detect autoantibodies directed against endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in the serum of GCA patients and to identify their target antigens Methods: Sera from 15 GCA patients were tested in pools of patients’ sera and compared to a sera pool from 12 healthy controls (HCs) Serum immunoglobulin G (IgG) reactivity was analysed by 2-D electrophoresis and immunoblotting with antigens from human umbilical vein ECs (HUVECs) and mammary artery VSMCs Target antigens were identified by mass spectrometry Results: Serum IgG from GCA patients recognised 162 ± (mean ± SD) and 100 ± 17 (mean ± SD) protein spots from HUVECs and VSMCs, respectively, and that from HCs recognised 79 and 94 protein spots, respectively In total, 30 spots from HUVECs and 19 from VSMCs were recognised by at least two-thirds and three-fifths, respectively, of the pools of sera from GCA patients and not by sera from HCs Among identified proteins, we found vinculin, lamin A/C, voltage-dependent anion-selective channel protein 2, annexin V and other proteins involved in cell energy metabolism and key cellular pathways Ingenuity pathway analysis revealed that most identified target antigens interacted with growth factor receptor-bound protein Conclusions: IgG antibodies to proteins in the proteome of ECs and VSMCs are present in the sera of GCA patients and recognise cellular targets that play key roles in cell biology and maintenance of homeostasis Their potential pathogenic role remains to be determined Introduction Giant cell arteritis (GCA), also known as temporal arteritis, is a primary systemic vasculitis involving large- and medium-sized vessels GCA commonly causes bitemporal headaches, jaw claudication, scalp tenderness and/ or abnormal temporal arteries (tender, nodular, swollen and thickened arteries with decreased pulses) detected during physical examinations GCA does not occur in * Correspondence: luc.mouthon@cch.aphp.fr † Contributed equally Inserm U1016, Institut Cochin, CNRS UMR 8104, rue Méchain, F-75014 Paris, France Full list of author information is available at the end of the article people younger than 50 years old, and its incidence increases with age and peaks in Caucasians older than 70 years of age [1,2] Ocular ischaemic complications occur in 25% of the patients and leads to irreversible visual loss in 15% [3] No definite immunological marker has been identified in GCA, and patients usually present with increased erythrocyte sedimentation rates and/or C-reactive protein levels Diagnosing GCA can be difficult, and temporal artery biopsy is the gold standard for making the diagnosis [4] However, in 10% to 20% of patients with GCA, the biopsy shows no specific change [5] © 2011 Régent et al.; licensee BioMed Central Ltd This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Régent et al Arthritis Research & Therapy 2011, 13:R107 http://arthritis-research.com/content/13/3/R107 GCA is an inflammatory condition of unknown origin characterised by the presence of giant cells and a remodelling process in the arterial wall [6] In patients with GCA, an immune-mediated reaction is suspected to be triggered by an antigen of unknown origin, either microbial or a self-antigen, that could be presented to T cells by dendritic cells [7] Thus, macrophages and giant cells stimulated by interferon-g (IFN-g) play a major role in the disruption of the elastic lamina and the remodelling of vessel walls In addition, in the adventitia, macrophages produce proinflammatory cytokines such as interleukin (IL-1) and IL-6, whereas in the media and intima they contribute to arterial injury by producing metalloproteinases and nitric oxide [6,8,9] Anti-endothelial cell (anti-EC) antibodies (AECAs) have been detected in a wide range of systemic inflammatory and/or autoimmune diseases, including primary and/or secondary systemic vasculitis [10] Although the pathogenic role of AECAs remains controversial [11,12], these antibodies may be responsible for EC activation [13] and induction of antibody-dependent, cell-mediated cytotoxicity and apoptosis [14] In GCA, AECAs were detected in 33% of sera by performing ELISA on fixed human umbilical vein ECs (HUVECs) [15], but their presence was not confirmed by indirect immunofluorescence [16] Anti-vascular smooth muscle cell (antiVSMC) antibodies have been detected in an experimental rat model of vasculitis [17]; however, to our knowledge, these antibodies have not been investigated in patients with primary systemic vasculitis We used 1-D and 2-D immunoblotting, followed by mass spectrometry (MS), to investigate the presence of autoantibodies directed against ECs and VSMCs and identify their target antigens in patients with GCA Materials and methods Patients Serum samples were obtained from 15 patients who fulfilled the American College of Rheumatology (ACR) criteria for GCA [4] and 33 patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis who fulfilled the ACR and the Chapel Hill criteria used as vasculitis controls, with the control group comprising 15 patients with Wegener’s granulomatosis (WG), with Churg-Strauss syndrome (CSS) and with microscopic polyangiitis (MPA) [18] In each group of patients with ANCA-associated vasculitis, two-thirds of the patients had active disease as assessed by a Birmingham Vasculitis Activity Score (BVAS) >3 in the absence of treatment, and one-third of the patients had inactive disease as assessed by a BVAS